We studied mechanisms for T cell recognition of antigens in association with major histocompatibility complex (MHC)-encoded molecules, and applications to the design of synthetic vaccines for AIDS and cancer. We have been characterizing the helper and cytotoxic T lymphocyte (CTL) responses to HIV envelope and reverse transcriptase, mapping the key epitopes, and defining the role of individual residues in these epitopes to be able to modify the structures to make more potent immunogens as vaccines. We have made vaccine constructs in which clusters of helper epitopes are synthesized coupled to a peptide that is a CTL epitope presented promiscuously by multiple class I MHC molecules in the human and mouse as well as a neutralizing antibody epitope. These constructs can induce all three arms of the immune response, neutralizing antibodies, CTL, and Th1 helper cells. Results of the first arm of a phase I clinical trial with one of these peptides show ability to induce CTL, helper T cell responses, and neutralizing antibodies to HIV in at least a subset of human recipients. Currently, we are developing new approaches in mouse models to develop second generation vaccine constructs. We have shown proof of principle that we can modify the sequence of a helper epitope of HIV to make it more immunogenic and also much more potent, when coupled to a CTL epitope, in eliciting CTL and protecting against viral infection. The enhanced helper epitopes elicit a stronger Th1 response and upregulate CD40L on the helper cells, which in turn induce more IL-12 production by dendritic cells, which then polarize the T helper cells to Th1. We are applying this "epitope enhancement" approach to conserved HIV helper and CTL epitopes from env, gag, and pol, presented by human class II and class I HLA molecules, as well as to hepatitis C virus (HCV) epitopes presented by human HLA-A2.1 (see below). We have developed an enhanced HIV reverse transcriptase epitope which is the subject of a clnical trial to be carried out with Dr. Robert Yarchoan, HAMB, CCR, NCI, and we have characterized human responses to an envelope helper epitope, which we have also enhanced. We have discovered ways of increasing CTL, helper, and antibody responses and steering them toward desired phenotypes, such as Th1 or Th2 or particular antibody isotypes, by incorporating cytokines into the emulsion adjuvant with the antigen. We compared a panel of 8 cytokines for their effects on 8 types of immune response, and discovered a novel synergy between GM-CSF and IL-12 and between TNF and IL-12 in induction of CTL. We found that all 3 cytokines provide triple synergy for induction of CTL with a peptide vaccine, for induction of interferon-gamma, and for protection against viral challenge in vivo, which we show to be interferon-gamma dependent. The mechanism of this synergy appears to relate to the upregulation of antigen presenting function by GM-CSF and of the IL-12 receptor by TNF and IL-12. Further, we have recently shown that GM-CSF that recruits dendritic cells and CD40L that matures them will synergize as a potent vaccine adjuvant combination for induction of CTL and protection against viral infection. We have even further enhanced this combination by a push-pull approach in which this potent combination of cytokine and costimulatory molecule is complemented by blocking a suppressive pathway with an inhibitor of IL-13, optimizing the vaccine-induced CTL response and protection. We have shown that high avidity CTL specific for HIV-1 envelope peptide are much more effective at clearing a recombinant vaccinia virus expressing HIV gp160 from SCID mice than are low avidity CTL specific for the same peptide-MHC complex, and have worked out two complementary mechanisms involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced, and to lyse targets more quickly. We have finally developed a method to preferentially elicit higher avidity CTL with a vaccine, by using costimulatory molecules to allow a response at lower antigen dose. However, we found that high avidity CTL are exquisitely sensitive to high dose antigen and will undergo programmed cell death, mediated by TNF and the TNF receptor II, but also requiring a permissive state involving a decrease in Bcl-2, IAP1, and TRAF2, and correlating with downmodulation of the T cell receptor. This effect may explain clonal exhaustion in viral infections. We have shown for the first time that protection against mucosal transmission of virus can be mediated by CD8 CTL without antibodies, but requires that the CTL be present at the mucosal site of transmission, whereas systemic CTL are not sufficient. The protection can be accomplished by intrarectal immunization with a peptide vaccine and increased by inclusion of IL-12 and GM-CSF with the vaccine. We found that endogenous IL-12 is less inhibited by the mucosal adjuvant LT(R192G) than by cholera toxin, and substituting this, the mucosal CTL response and protection are less dependent on exogenous IL-12. Using this mutant LT, we immunized MamuA*01-positive Rhesus macaques intrarectally with a similar peptide AIDS vaccine and induced CTL in the colon and mesenteric lymph nodes that have impacted the clearance of virus after intrarectal challenge with pathogenic AIDS virus SHIV-Ku. Intrarectal immunization was more effective than subcutaneous immunization with the same peptide vaccine at protecting against SHIV, in part because we found the induction of mucosal CTL provided for greater clearance from a major site of virus replication, the gut mucosa, which was seeding the bloodstream. We have commenced a second Rhesus macaque mucosal vaccine study using mucosal peptide priming, with a combination of cytokines as adjuvants, and using mucosal boosting with a recombinant poxvirus, to prevent AIDS virus transmission across a mucosal barrier. With regard to cancer, we identified several CTL epitopes in proteins of hepatitis C virus (HCV), that causes liver cancer, using a novel approach, and have analyzed the role of each amino acid residue in order to modify one of the peptides to make a more potent vaccine. Using this epitope enhancement approach, we could increase the immunogenicity of an epitope of the HCV core protein, presented by the most common human class I HLA molecule, HLA-A2.1, both for HLA-A2.1-transgenic mice in vivo and for human T cells in vitro. This enhanced epitope is being incorporated into a vaccine. We are attempting to enhance other HCV core epitopes to incorporate into a DNA vaccine. We also developed a model of immunosurveillance of cancer in which tumors are rejected by CD8 T cells, but the rejection is incomplete in the presence of normal CD4 regulatory cells, and an escape variant of the tumor recurs. However, depletion of CD4 cells allows complete eradication of the tumor by CD8 cells. Using receptor knock-out mice, we found that the key regulatory cytokine inhibiting immunosurveillance against cancer was IL-13, acting through the IL-4 receptor/STAT6 pathway, although IL-4 itself was neither necessary nor sufficient. We discovered that the major source of IL-13 was NKT cells, and that absence of these in CD1-knockout mice prevented tumor recurrence in these mice. We have now found that this regulatory pathway applies also to murine models of breast cancer, colon cancer, and osteosarcoma, and that in each case we can prevent tumor growth by abrogating this immunoregulatory pathway. Blockade of IL-13 also delayed spontaneous breast cancer formation in Her-2/neu-transgenic mice. We are also determining the mechanism by which IL-13 indirectly inhibits CD8 T cell-mediated immunosurveillance when the CD8 T cells do not have IL-13 receptors. We have identified an intermediate non-lymphoid cell that responds to IL-13 and then suppresses the anti-tumor CTL response. We are also developing clinical trial approaches to amplify immunotherapy of cancer by inhibiting IL-13. We developed peptide cancer vaccines inducing CTL immunity to mutant p53 expressed in cancer cells. We found that mutant p53 peptides, coated on dendritic cells, elicit CTL that kill tumor cells expressing the mutation and suppress established tumors in animals. Common mutations in ras peptides were found to enhance binding to HLA-A2.1, but also to influence antigen processing. We are applying epitope enhancement to the mutant ras peptides to make more effective vaccines. We also induced murine CTL against fusion proteins from chromosomal translocations in pediatric tumors, alveolar rhabdomyosarcoma and Ewing's sarcoma. We also identified novel epitopes spanning these fusion protein junctions in these sarcomas, synovial sarcoma, and others, that could bind to several human HLA molecules, HLA-A1, A3, B7 and B27, and were able to map a minimal epitope in synovial sarcoma presented by HLA-B7 and elicit human CTL that could kill human sarcoma tumor cells, proving that these fusion proteins are promising tumor antigens for cancer immunotherapy. Recently we have defined epitopes in two newly discovered prostate cancer antigens, TARP and XAGE1, in collaboration with Ira Pastan (CCR, NCI), and have obtained HLA-A2-restricted T cell responses in HLA- transgenic mice. 29 patients were treated in a phase I/II clinical trial of the mutant p53/ras peptide vaccine approach to treating cancer, and a large fraction have made CTL or cytokine responses, and no adverse effects were seen. Importantly, we found that a cellular immune response to the mutant peptide was statistically significantly associated with longer survival. A trial of translocation fusion peptide immunization of patients with alveolar rhabdomyosarcoma and Ewing's sarcoma is underway. We have also started a trial of immunization of cervical cancer patients with peptides from the E6 and E7 oncoproteins of human papillomavirus type 16 that bind to HLA-A2.1 in patients who express this HLA molecule. Many patients made CTL responses, and some had unexpectedly stable disease. A phase II trial of autologous dendritic cells pulsed with mutant ras peptides corresponding to the patient's tumor in colon cancer patients with HLA-A2.1 that can present these ras peptides is underway. This protocol and a new one to treat non-small cell lung cancer patients with autologous mutant p53 peptide-pulsed dendritic cells have been modified to mature the dendritic cells with CD40 ligand, which has recently been made available. This should greatly improve immunogenicity of the peptide-dendritic cell vaccine. The second patient treated had a CTL response and a mixed clinical response. (50% AIDS related)